I purified my membrane protein by FPLC gel filtration column. Before the injection , I checked the OD280, which is around 1AU. then i took 0.5ml protein sample to run the FPLC. However, no sharp peak came out. I just got a broad and small peak with 35 mAu. which is much lower than that expected. (I purified the membrane protein with DM and the FPLC buffer : 150mM NaCl, 20mM HEPEs, 0.4g/100ml DM)
Is it that my protein has aggregated on the FPLC column?
I also try to increase salt concentration (300mM) of the FPLC buffer , then I got a sharp peak with OD 160 mAu and the retention time is some early than Void Volume. Is it normal ? anybody can give me some suggestion for the FPLC pufication condition?
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mdfenko
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Posted 02 March 2010 - 12:57 PM
which fplc column did you use?
recommended minimum ionic strength for fplc buffer (gel filtration) is 200mM. this prevents non-specific binding to the stationary phase.
if the peak came before the void then it was something released from the column prior to the injection. when you re-equilibrated with the higher salt buffer you probably did not completely wash the column before you injected (anything bound that was released will fractionate).
recommended minimum ionic strength for fplc buffer (gel filtration) is 200mM. this prevents non-specific binding to the stationary phase.
if the peak came before the void then it was something released from the column prior to the injection. when you re-equilibrated with the higher salt buffer you probably did not completely wash the column before you injected (anything bound that was released will fractionate).
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